Diversity of Bacterial Clones and Plasmids of NDM-1 Producing Escherichia coli Clinical Isolates in Central Greece

The objective of the present study was to genetically characterize ten NDM-1 producing Escherichia coli isolates, recovered from patients in a hospital in Central Greece during the period 2017 to 2021.The isolates were studied by whole genome sequencing to obtain multi-locus sequencing typing (MLST), identification of blaNDM1-environment, resistome and plasmid content. MLST analysis showed the presence of eight sequence types: ST46* (two isolates), ST46, ST744, ST998, ST410, ST224, ST4380, ST683 and ST12 (one isolate each). Apart of the presence of blaNDM-1, the isolates carried a combination of various to β-lactams encoding resistance genes: blaTEM-1B, blaCTX-15, blaOXA-1, blaVIM-1, blaSHV-5, blaOXA-16, blaOXA-10 and blaVEB-1. Additionally, plurality of resistance genes to aminoglycosides, macrolides, rifamycin, phenicols, sulfonamides and tetracycline was detected. The presence of multiple replicons was observed, with predominance of IncFII and IncFIB. Analysis of blaNDM-1 genetic environment of the isolates showed that seven had 100% identity with the pS-3002cz plasmid (Accession Number KJ 958927), two with the pB-3002cz plasmid (Accession Number KJ958926) and one with the pEc19397-131 plasmid (Accession Number MG878866). Τhis latter plasmid was derived by the fusion of two, previously identified, plasmids, pAMPD2 and pLK75 (Accession Numbers CP078058 and KJ440076, respectively). The diversity of clones and plasmids of NDM-1 producing E. coli isolated from patients in Greece indicates a continuous horizontal gene transfer.


Introduction
Escherichia coli is a bacterial species with high diversity, ranging from intestinal commensal strains to pathogenic strains causing urinary tract infection, acute enteritis, sepsis and neonatal meningitis [1]. In recent years, carbapenem-resistant E. coli (CREC) has been a serious problem worldwide, attracting significant clinical interest and attention [2,3]. As in carbapenem-resistant Enterobacterales, the acquisition of carbapenemase-encoding genes is also the most common mechanisms for CREC [3,4].
Recently, several reports have demonstrated the dissemination of bla NDM encoding gene among E. coli isolated from patients in various geographical locations [7]. The heterogenicity of the genetic environment of bla NDM encoding gene, combined with its location on plasmids of different incompatibility groups (Inc) has led to the emergence of diverse CREC clones worldwide [8,9].
In Greece, carbapenem-resistant E. coli isolates have been recovered rarely [10]. Two previous studies have reported the presence of bla KPC , bla NDM-1 and bla NDM-5 positive E. coli isolates, recovered from humans and animals [10,11]. Although clonality was 2 of 7 observed among bla KPC positive E. coli, primary data have revealed a diversity of clones among NDM-producing E. coli [10]. This finding has raised the questions, first if the same or different plasmids have been introduced into E. coli isolates, and second if the genetic context of bla NDM-1 would be the same among the various clones.
The objective of the present study was to genetically characterize ten NDM-producing E. coli isolates, recovered from patients in Central Greece during the period 2017 to 2021.

Isolation of bla NDM-1 Escerichia coli
Ten (10) bla NDM-1 positive E. coli isolates were studied in this work. All isolates had been recovered from clinical samples of patients admitted to the University Hospital of Larissa in Central Greece, a tertiary-care hospital, during the period 2017 to 2021.
The identification and the susceptibility testing of the isolates were carried out using the automated Vitek-2 system (BioMerieux, Marcy l' Etoile, France). Minimal Inhibitory Concentrations (MICs) of imipenem and meropenem were determined by MIC test strip (Liofilchem, Roseto degli Abruzzi, Italy); MIC to colistin was determined by broth microdilution method, following the respective EUCAST guidelines (https://www.eucast.org, accessed on 10 October 2022). All the isolates were tested for phenotypic production of carbapenemase, by using MIC strips containing meropenem plus ethylenediaminetetraacetic acid (EDTA) and meropenem plus phenylboronic acid (Liofilchem).
Isolates found with a ratio meropenem/(meropenem plus EDTA) ≥ 8 were subsequently tested for detection of the carbapenemase encoding genes bla VIM , bla NDM , by means of a relevant PCR followed by sequencing analysis [10]. The ten (10) bla NDM-1 positive E. coli were identified and were further studied by Whole Genome Sequencing (WGS) analysis.

Whole Genome Sequencing of bla NDM-1 Positive E. coli
Initially, libraries were prepared using Ion Torrent technology and Ion Chef work flows (Thermo Fisher Scientific, Waltham, MA, USA). Genomic DNA libraries were sequenced on the S5XLS system and analysis of primary data was conducted with Ion Torrent Suite v.5.10.0 (Thermo Fisher Scientific). The quality of the reads was checked using FastQC software version 0.11.9. The reads for each sample were assembled using the SPAdes genome assembler v3.15.5 with the default parameters. The quality of the assembled genomes was assessed with the tool Quast version 5.2.0. The average coverage for each genome was computed using the tool mapPacBio from BBTools (https://sourceforge. net/projects/bbmap/, accessed on 25 October 2022). Gaps were filled by sequencing of overlapping PCR produced fragments.
Typing of isolates, based on the Achtman scheme, was assessed by using the online tool MLST 2.0. Evaluation for the presence of antibiotic resistance genes in the assembled genomes was performed by using the online tool ResFinder-4.1, with the ID threshold set to 90% and the minimum length set to 60%. The presence of plasmids in the assemblies was assessed by means of Plasmid Finder v2.1, using the Enterobacterales database, with the minimum identity threshold set to 95% and the minimum coverage set to 60%. In order to determine the origin of the genetic contexts of bla NDM-1 encoding gene, a BLAST analysis was performed. Only results with a high identity score (100% identity and ≥90% cover age) were considered.

Plasmid Analysis of bla NDM-1 Klebsiella Pneumoniae Strains
The bla NDM-1 encoding plasmids identified in E. coli isolates were compared to plasmids found among K. pneumoniae population. Five bla NDM-1 positive K. pneumoniae strains, all of them recovered during the study period (2017-2021) at the University Hospital of Larissa, were also selected for plasmid analysis. Selection was based on their ST types and the presence of carbapenemase-encoding genes. Two of these strains (B3118 and B3119), belonging to ST11, carried both bla NDM-1 and bla OXA-48 encoding genes, one strain (B3185), belonging to ST11, carried both bla NDM-1 and bla KPC encoding genes, whilst the other two strains, belonging to ST11 (B3173) and ST231 (B3214), carried only the bla NDM-1 encoding gene. Whole genome analysis of those strains had been performed as described above (Section 2.2).

Data Management
Data were entered into Microsoft Excel and analysed using SPSS v. 26 (IBM Analytics, Armonk, NY, USA). Initially, descriptive analysis was performed. Then, comparisons were performed by employing Pearson's chi-square or Fisher exact test, as appropriate, and analysis of variance, according to the type of data. Statistical significance was defined at p < 0.05.

Multi-Locus Sequence Typing (MLST)
In silico performed MLST revealed that the ten isolates belonged to nine distinct Sequence Types (STs), specifically one isolate in each of ST744, ST998, ST410, ST224, ST4380, ST46, ST683, ST12, whilst two isolates were found to belong to a new type. This included a one base difference from adk 840 allele ( 535 C → T), while the alleles of the other six genes were identical to those of ST46; it is noted as ST46*.

Identification of Resistance Genes
WGS analysis revealed that the ten bla NDM-1 positive E. coli strains cumulatively possessed 40 distinct resistance genes. Cumulatively, the ten strains possessed nine distinct genes encoding resistance to β-lactams and 13 distinct genes encoding resistance to aminoglycosides (i.e., 58.5% of distinct genes found, encoded resistance against one of these two antibiotic classes).
Median number of genes per strain was 14 (min.: 8 -max.: 20). Apart from bla NDM-1 , no other β-lactamase encoding gene was detected in all ten strains. Other frequently detected genes were aph(3")-Ib, aph(6)-Id and sul2 (in eight strains each), as well as bla TEM-1B and tetA (in seven strains each). Nine strains carried at least three genes encoding resistance to β-lactams (median number of such genes per strain = 3) and eight of these at least three genes encoding resistance to aminoglycosides (median number of such genes per strain = 4). These higher per strain numbers of genes encoding resistance against βlactams or aminoglycosides were significant (p < 0.0001).
All strains carried genes conferring resistance to at least six antimicrobial agents, whilst three strains carried genes against eight antimicrobial agents. A summary of the genes identified in the ten strains is in Figure 1. sistance to β-lactams (median number of such genes per strain = 3) and eight of these at least three genes encoding resistance to aminoglycosides (median number of such genes per strain = 4). These higher per strain numbers of genes encoding resistance against βlactams or aminoglycosides were significant (p < 0.0001).
All strains carried genes conferring resistance to at least six antimicrobial agents, whilst three strains carried genes against eight antimicrobial agents. A summary of the genes identified in the ten strains is in Figure 1.

Identification of Plasmids
Analysis of replicon sequences revealed that the ten strains carried 20 types of plasmids. Among these, IncFIB predominated (in eight strains), followed by IncI1-I(Alpha) and IncC (in seven strains each); IncFII was also frequently detected in five strains.
Median number of plasmids per strain was 6 (min.: 3 -max.: 9). In nine strains, at least four and in seven strains at least six replicons were identified.
Finally, three distinct Col plasmids were also detected. A summary of the replicons identified in the ten strains is in Figure 1.

Identification of Plasmids
Analysis of replicon sequences revealed that the ten strains carried 20 types of plasmids. Among these, IncFIB predominated (in eight strains), followed by IncI1-I(Alpha) and IncC (in seven strains each); IncFII was also frequently detected in five strains.
Median number of plasmids per strain was 6 (min.: 3-max.: 9). In nine strains, at least four and in seven strains at least six replicons were identified.
Finally, three distinct Col plasmids were also detected. A summary of the replicons identified in the ten strains is in Figure 1.

Discussion
The first description of blaNDM-1 was published in 2009 [14]. K. pneumoniae and E. coli are the most commonly described NDM-1 producing bacteria. However, the blaNDM-1 encoding gene has also been detected in Enterobacterales other than K. pneumoniae and E. coli [15]; NDM-1 production has been also found in clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa, as well as in a wide variety of other, non-fermenting Gramnegative bacteria [16].
Until 2017, no blaNDM-1 positive E. coli isolates had been reported from Greece; in contrast, several studies had indicated the dissemination of blaNDM-1 positive K. pneumoniae in many hospitals of the country; in a large multicenter study, conducted between 2013 to 2016 across the country, 71% of K. pneumoniae isolates found to be phenotypically MBL positive, were also found to be blaNDM-1 positive, with ST11 being the predominant clone [17][18][19][20]. The same epidemiological findings, with little differences regarding blaNDM-1 positive K. pneumoniae frequency and clonality, are observed up today in the country.
The first three blaNDM-1 positive E. coli strains in the country were recognized in 2017 at the University Hospital of Larissa, a tertiary care hospital located in Central Greece [10]. During the following years (2018-2021), another seven isolates have been recognized from clinical specimens collected from patients in the hospital. The proportion of blaNDM-1 positive E. coli has remained stable throughout the study period, corresponding to 0.17% of total E. coli isolates recovered annually.

Discussion
The first description of bla NDM-1 was published in 2009 [14]. K. pneumoniae and E. coli are the most commonly described NDM-1 producing bacteria. However, the bla NDM-1 encoding gene has also been detected in Enterobacterales other than K. pneumoniae and E. coli [15]; NDM-1 production has been also found in clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa, as well as in a wide variety of other, non-fermenting Gram-negative bacteria [16].
Until 2017, no bla NDM-1 positive E. coli isolates had been reported from Greece; in contrast, several studies had indicated the dissemination of bla NDM-1 positive K. pneumoniae in many hospitals of the country; in a large multicenter study, conducted between 2013 to 2016 across the country, 71% of K. pneumoniae isolates found to be phenotypically MBL positive, were also found to be bla NDM-1 positive, with ST11 being the predominant clone [17][18][19][20]. The same epidemiological findings, with little differences regarding bla NDM-1 positive K. pneumoniae frequency and clonality, are observed up today in the country.
The first three bla NDM-1 positive E. coli strains in the country were recognized in 2017 at the University Hospital of Larissa, a tertiary care hospital located in Central Greece [10]. During the following years (2018-2021), another seven isolates have been recognized from clinical specimens collected from patients in the hospital. The proportion of bla NDM-1 positive E. coli has remained stable throughout the study period, corresponding to 0.17% of total E. coli isolates recovered annually.
The dissemination of bla NDM-1 encoding gene among K. pneumoniae and E. coli is carried out via plasmids or other mobile genetic elements. Previous studies have shown the presence of two plasmids, pB-3002cz and pS-3002cz, both previously identified in the Czech Republic, among the bla NDM-1 positive K. pneumoniae strains isolated in the country [17][18][19]; however, no data have been reported about the characterization of plasmids among bla NDM-1 E. coli. The above two plasmids were carried by nine of the bla NDM-1 positive E. coli studied in the present work. Given that the bla NDM-1 positive K. pneumoniae previously recovered, also carried the same plasmids, the present findings indicate bla NDM encoding gene' transmission between different bacterial species and different clones of the same species.
Only one bla NDM-1 positive E. coli studied in the present work, was found to carry an uncommon plasmid, previously found in a strain recovered in China in 2018. Clinical